Project Summary The Hedgehog (Hh) signaling pathway plays a central role in specifying the embryonic pattern of metazoan organs. Post-embryonically, the Hh pathway controls homeostatic maintenance and injury-induced regeneration of adult tissues. Drugs that block Hh pathway activity have received FDA approval or are in clinical trials for treatment of basal cell carcinoma or medulloblastoma, cancers that are associated with elevated pathway activity in primary tumor cells. In pancreatic, bladder, and other cancers of endodermal origin, Hh pathway activity in tumor-associated stroma restrains cancer growth and progression, suggesting pharmacological pathway activation as a potential therapeutic approach. In addition, pathway activation may have a therapeutic role in regenerative medicine through its ability to enhance neurogenesis (e.g., in Down Syndrome), to accelerate bone and muscle repair, and to ameliorate inflammatory bowel disease, among other emerging biological activities. Despite the importance of Hh signaling in development and physiology, and the established and growing role of pathway modulation in control of malignant and non-malignant disease, we fundamentally do not understand how the extracellular Hh protein signal is transduced across the membrane. The Hh receptor is unusual in that its signaling activity is subdivided into sensing of the extracellular Hh ligand by the transporter-like protein Patched (Ptch), and propagation of this signal into the cell by the seven- transmembrane protein Smoothened (Smo). Mechanistically, Ptch is thought to inhibit Smo by acting as a chemiosmotically-driven transmembrane transporter of a lipidic Smo modulator, but the chemiosmotic driver and lipidic substrate of Ptch remain to be elucidated, as does the mechanism by which Hh binding disrupts Ptch activity. We have developed an in vitro assay that directly monitors activity and conformation of a purified, reconstituted Smo protein allowing us to directly test Smo modulation by distinct classes of ligands, including a recently identified class that is the leading candidate for mediating Ptch suppression of Smo. We also have developed a real-time in vivo assay of Smo conformation that monitors Ptch activity, and will use this assay to further elucidate the role of a candidate chemiosmotic driver that we have recently identified. Furthermore, we have purified an active, well-behaved form of Ptch for functional reconstitution and for tests of interaction with candidate mediators; we have in addition generated a cryo-EM based structure of this Ptch protein in complex with a Hh ligand, and will refine this structure as a basis for illuminating the mechanism of Ptch-Smo regulation and understanding Hh inactivation of Ptch. Finally, we will determine how the dual lipid adducts of the mature Hh ligand are shielded within the soluble complex that allows movement of Hh from its cellular source to the Ptch receptor on target cells, and how the transition from this complex to interaction with Ptch is accomplished. Our findings will be integrated into a detailed molecular account of Hh signal transduction, and may provide a basis for improvements in cancer or regenerative therapies involving Hh pathway inhibition or activation.